[unreadable] [unreadable] We have invented a safe and simple method for naked DNA delivery into limb muscles, the hydrodynamic limb vein injection, which is a clinically viable procedure holding great promise for the treatment of various inherited and acquired diseases. This technology increased the efficiency of naked DNA delivery to a level that makes its transfer to the clinic realistic. However, a significant increase in expression levels would make treatments more economical and would also make the approach amenable for a broader range of disorders. For this Phase I project we propose to test the use of the combination of two promoters that we found to interact in trans in a synergistic way in rhesus skeletal muscle leading to over 10-fold enhancement in gene expression. As the first steps toward characterizing the phenomenon and identifying the mechanism behind it our specific aims will assess whether the synergistic effect: (1) can be reproduced in several species commonly used for in vivo gene delivery studies, (2) requires the presence of the promoters in trans, or new constructs containing the promoters in cis on a single plasmid would also show enhanced expression, (3) can sustain elevated expression levels long-term, (4) increases the percentage of myofibers with detectable (therapeutic) level of transgene expression besides increasing the overall amount of gene product. Our long- term goals for Phase II studies are to identify the sequence elements that are responsible for the synergistic effect and the mechanism by which they interact and use the information for the rational design of new expression constructs. A 10-fold increase in expression efficiency would have a remarkable effect on how economical naked DNA-based gene therapy may become. It would facilitate the transition of many current non-viral gene therapy research projects to the clinic. There are a great number of inherited and acquired diseases that could benefit from gene therapy. The intravenous injection of naked DNA into limb muscles has the potential to become a clinically acceptable gene delivery procedure. We plan to evaluate a unique approach to further improve expression efficiency in order to make this technology applicable for a broader range of diseases. [unreadable] [unreadable] [unreadable] [unreadable]